Production Of BFGF Monoclonal Antibody And Its Inhibition Of Lewis’ Lung Carcinoma

Objective: To produce mouse monoclonal antibody(Mc Ab) against basic fibroblast growth factor(b FGF)of human directly at the binding site of b FGF with FGFR1 and then preliminarily study its anti-tumor activity and mechanism of Lewis’ lung carcinoma.Methods: BALB/c mice were immunized with recombinant human b FGF. Spleen lymphocytes were fused with SP2/0 mouse myeloma cells. Hybridoma cell lines secreting Mc Ab against the binding site of b FGF with FGFR1ⅢC were screened out by indirect and competitive ELISA. The Mc Ab in ascites was purified by using affinity chromatography with Protein G and then identified by SDS-PAGE. ELISA was further carried out to determine the antibody isotype、affinity constant and IC50.Gene of Mc Ab Fv fragment was cloned by RT-PCR, then compared the binding sites of Fv-b FGF modeling by Discovery Studio 4.0 with that of FGFR1-b FGF. The effect of anti-b FGF Mc Ab on proliferation inhibition and migration of Lewis’ cell was measured by CCK-8 and transwell assay respectively. C57BL/6 mouses were injected with Lewis’ cells subcutaneously, and then were cured with anti-b FGF Mc Ab by injecting around tumor. Measured the tumor size and counted the lung metastatic focis of mouses. The expression level of E-cadherin、phosphorylated c-src and β-catenin in Lewis’ cell were detected by real-time PCR and western blot after cultivating with anti-b FGF Mc Ab.Results: Two hybridoma cell strains named MabE12 and MabD9 stably secreted anti-bFGF monoclonal antibody aiming at the binding site of b FGF-FGFR1βⅢC were obtained. Competitive ELISA showed the IC50 of Mab E12 and Mab D9 were 1.564μg/m L and 1.96μg/m L respectively and they were both belonged to Ig G1. Mab E12, selected as the following studying material had a relative affinity constant as 5.66×108L/mol. Furthermore, Mab E12 could specifically react with nature b FGF expressed in Lewis’ cells by western blot and immunofluorescence analysis. The result of CCK-8 assay showed Mab E12 had a good effect on the proliferation inhibition of Lewis’ cell with IC50 as 41μg/m L and also it could significantly inhibit migration of Lewis’ cell(P<0.05). Although the result of in vivo anti-tumor study showed Mab E12 could not inhibit tumor growth significantly compared with control(P=0.056), it could up-regulate expression of E-cadherin(P<0.05). Further studying result indicated Mab E12 up-regulate the expression of E-cadherin(P<0.05)and the level of phosphrylated c-src、β-catenin.Conclusion: Two hybridoma cell strains stably secreting McAb that can specifically recognize the binding site of b FGF with FGFR1βⅢC were developed successfully, and one of them named Mab E12, can up-regulate the expression of E-cadherin in Lewis’ cells and effectively inhibit migration of Lewis’ cells in vitro. The mechanism of this phenomenon may lie in the up-regulation of phosphorylated c-src and β-catenin by Mab E12.